Method for manufacturing protein bioelectronic devices

Abstract

The present disclosure provides devices, systems, and methods related to protein bioelectronics. In particular, the present disclosure provides devices, systems, and methods for forming electrical contacts to a protein with high yield, which facilitates the manufacture of analytical devices to detect and measure the electrical characteristics corresponding to protein function.

What is claimed is: 
     
         1 . A method of manufacturing a device for direct measurement of protein activity, the method comprising:
 combining a first and second electrode with a protein-of-interest to form an electrical connection between the electrodes, wherein the first and second electrodes comprise surfaces chemically modified with a linker molecule, and wherein the protein-of-interest comprises at least one non-canonical amino acid;   wherein applying a voltage bias to the electrodes produces current flow through the protein-of-interest.   
     
     
         2 . The method of  claim 1 , wherein fluctuations in activity of the protein-of-interest correspond to fluctuations in current. 
     
     
         3 . The method of  claim 1 , wherein the surfaces of the first and second electrodes are chemically modified with at least one thiolated biotin linker molecule. 
     
     
         4 . The method of  claim 1 , wherein the at least one non-canonical amino acid comprises biotin or a derivative thereof. 
     
     
         5 . The method of  claim 4 , wherein the at least one non-canonical amino acid is biocytin or a derivative thereof. 
     
     
         6 . The method of  claim 4 , wherein the protein-of-interest comprises two biocytin non-canonical amino acids or derivatives thereof. 
     
     
         7 . The method of  claim 1 , wherein the protein-of-interest comprises an Avitag sequence or a derivative thereof. 
     
     
         8 . The method of  claim 1 , wherein the protein-of-interest does not comprise an Avitag sequence or a derivative thereof. 
     
     
         9 . The method of  claim 1 , wherein the method further comprises adding a second linker molecule to form the electrical connection. 
     
     
         10 . The method of  claim 9 , wherein the second linker molecule comprises a streptavidin molecule. 
     
     
         11 . The method of  claim 10 , wherein the streptavidin molecule comprises at least two biotin binding sites. 
     
     
         12 . The method of  claim 1 , wherein the protein-of-interest comprises the least one non-canonical amino acid at two distinct locations. 
     
     
         13 . The method of  claim 12 , wherein the distinct locations comprise at least one of: (i) non-adjacent locations; (ii) locations that do not undergo substantial movement during protein activity; (iii) locations that are on an accessible surface of the protein-of-interest; and/or (iv) locations that are separated by at least 5 nm. 
     
     
         14 . The method of  claim 1 , wherein the protein-of-interest is selected from the group consisting of a polymerase, a nuclease, a proteasome, a glycopeptidase, a glycosidase, a kinase and an endonuclease. 
     
     
         15 . The method of  claim 1 , wherein the protein-of-interest is a polymerase. 
     
     
         16 . The method of  claim 15 , wherein exonuclease activity of the polymerase is disabled. 
     
     
         17 . A device for direct measurement of protein activity, the device comprising:
 a first electrode and a second electrode, wherein the first and second electrodes comprise surfaces chemically modified with at least one thiolated biotin linker molecule; and   a protein-of-interest that forms an electrical connection between the first and second electrodes comprising at least one non-canonical amino acid, wherein the at least one non-canonical amino acid comprises biotin or a derivative thereof;   wherein applying a voltage bias to the electrodes produces current flow through the protein-of-interest.   
     
     
         18 . The device of  claim 17 , wherein fluctuations in activity of the protein-of-interest correspond to fluctuations in current. 
     
     
         19 . The device of  claim 17 , wherein the at least one non-canonical amino acid is biocytin or a derivative thereof. 
     
     
         20 . The device of  claim 17 , wherein the protein-of-interest comprises two biocytin non-canonical amino acids or derivatives thereof. 
     
     
         21 . The device of  claim 17 , wherein the protein-of-interest comprises an Avitag sequence or a derivative thereof. 
     
     
         22 . The device of  claim 17 , wherein the protein-of-interest does not comprise an Avitag sequence or a derivative thereof. 
     
     
         23 . The device of  claim 17 , wherein the device further comprises a second linker molecule comprising a streptavidin molecule. 
     
     
         24 . The device of  claim 17 , wherein the protein-of-interest comprises the least one non-canonical amino acid at two distinct locations. 
     
     
         25 . The device of  claim 24 , wherein the distinct locations comprise at least one of: (i) non-adjacent locations; (ii) locations that do not undergo substantial movement during protein activity; (iii) locations that are on an accessible surface of the protein-of-interest; and/or (iv) locations that are separated by at least 5 nm. 
     
     
         26 . The device of  claim 17 , wherein the protein-of-interest is selected from the group consisting of a polymerase, a nuclease, a proteasome, a glycopeptidase, a glycosidase, a kinase and an endonuclease. 
     
     
         27 . The device of  claim 17 , wherein the protein-of-interest is a polymerase. 
     
     
         28 . The device of  claim 27 , wherein exonuclease activity of the polymerase is disabled. 
     
     
         29 . A system for direct electrical measurement of protein activity, the system comprising:
 the device of  claim 17 ;   a means for introducing a chemical entity that is capable of interacting with the protein-of-interest;   a means for applying a voltage bias between the first and second electrodes that is 100 mV or less; and   a means for monitoring fluctuations that occur as the chemical entity interacts with the protein-of-interest.